Viral infections are a principal cause of illness due to communicable diseases that affect the public at large. Of these, influenza viruses, including types A and B, are a significant factor responsible for causing respiratory symptoms as well as systemic malaise; other respiratory viruses include parainfluenza 1, 2, 3, and 4, respiratory syncytial virus, and adenovirus. The influenza viruses undergo rapid mutation of strains, producing pathogens with varying degrees of virulence and severity of symptoms. Recently, influenza infection has been as high as the fifth leading cause of death from acute respiratory disease in the United States (Morbidity and Mortality Weekly Report, 36 (1987) 2).
For this reason it is important to have available ways of diagnosing the presence of an influenza infection, and of distinguishing it from related viral and bacterial infections. Particularly among infants, the elderly and those having compromised or deficient immune responses, early diagnosis of influenza can lead to appropriate symptomatic treatment to minimize the risk of morbidity.
Diagnosis of viral infection, such as infection by influenza virus, may be carried out by detecting the presence of unique moieties characteristic of the virus. Virus particles typically carry distinctive antigenic components on the exterior of the virion which may be detected by specific ligand-antiligand interactions, in particular by the use of an antibody specific for a viral epitope. Such interactions rely on the law of mass action, and for this reason may have limited sensitivity. Many virus particles additionally carry specific enzymatic activities on the virion particle. Influenza virus is an example of such virus, endowed with a virus-specific neuraminidase activity as an integral part of the virion which is exposed to the environment. Utilization of the enzymatic activity in such cases offers the potential for increasing the sensitivity of a detection method.
N-acetylneuraminic acid (sialic acid, Neu5Ac), whose structure is shown below with atoms numbered, ##STR1##
is the terminal saccharide residue of many complex carbohydrate side chains of cell surface glycoproteins. In this structure the C2 position is the anomeric carbon, which is characterized by being part of a hemiketal group, or the 2-ketoside moiety. The glycosidic linkage bonding Neu5Ac with the penultimate saccharide is the substrate of the neuraminidase activity of the influenza virion. The neuraminidase hydrolyzes the linkage, thereby cleaving Neu5Ac from the penultimate saccharide. Consequently, suitable synthetic substrates may be derivatives of Neu5Ac in 2-ketoside linkage with a detectable moiety. The moiety then provides a product, when the substrate is acted upon by the viral neuraminidase activity, that signals the presence and amount of influenza virus particles in a sample. Since the viral enzyme cleaves the substrate catalytically, the sensitivity of detecting the presence of the enzyme is greatly enhanced. For this reason the overall sensitivity of detection may be comparable to, and may even be improved over, that provided by antibody binding assays.
One method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Pat. No. 5,252,458, which is incorporated herein by reference. An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al. (J. Infectious Diseases 142 (1980) 516-523). Yolken et al. used the 4-methylumbelliferyl-2-ketoside of Neu5Ac as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated virus as well as in some nasal wash specimens from human volunteers infected with the influenza virus. Yolken et al. suggested that "successful development of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventive and therapeutic interventions." According to Yolken et al., colorimetric assays were insufficiently sensitive for clinical applications, suggesting instead that fluorimetric assays for influenza neuraminidase might be suitable for detecting the virus in clinical samples.
Pachucki et al. (J. Clinical Microbiology 26 (1988) 2664-2666) tested the 4-methylumbelliferyl-2-ketoside of Neu5Ac on clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful in detecting neuraminidase in clinical specimens. The assay did, however, identify 91% of virus-positive isolates 25 hours after inoculation of tissue cultures.
The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, the C4 position has been reported to play an important role in enzyme-substrate interactions. Further, since it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem. 258 (1983) 12465-12471), it is essential to avoid these undesired enzymatic activities. It has, for example, been shown that ketosides of 4-methoxy-Neu5Ac are resistant towards certain bacterial sialidases (Beau et al., Eur. J. Biochem. 106 (1980) 531-540).
Although modification of the 4-position of Neu5Ac's provides specificity between certain viral and certain bacterial neuraminidase activities, it is still desirable to obtain substrates which provide enhanced specificity between the various viral neuraminidase reactivities while maintaining the specificity between viral and bacterial neuraminidase activities. Such substrates allow, for example, high specificity for particular types of neuraminidase-containing viruses and allow better and more directed treatment regimes. Use of specific substrates thus allows for more accurate surveillance of viral infections and more focused medical intervention as appropriate. U.S. Pat. No. 5,719,020, incorporated herein by reference, provides chromogenic and fluorogenic 4,7-disubstituted N-acetylneuraminic acid substrates for viral neuraminidase activities. These derivatives provide further specificity or differentiation between the various viral neuraminidase activities while maintaining the specificity between viral and bacterial neuraminidase activities. In particular, in U.S. Pat. No. 5,719,020 it is shown that the 4,7-modified Neu5Ac chromogenic substrates disclosed therein distinguish between influenza type A and B viruses, on the one hand, and neuraminidases from other viral and bacterial pathogens, on the other. A chromogenic derivative of 4-O-methyl Neu5Ac, however, develops color when exposed to parainfluenza virus types 1 and 2, and mumps, in addition to influenza types A and B.
The synthetic procedures leading to the 4,7-disubstituted chromogenic Neu5Ac ketoside derivatives disclosed in U.S. Pat. No. 5,719,020 have certain difficulties associated with them. These include the provision of synthetic routes involving a large number of distinct chemical reactions. These require an excessive number of unit operations, leading to the need for many reagents, solvents, pieces of laboratory apparatus, and for extended time to obtain the product. Certain reactions disclosed in U.S. Pat. No. 5,719,020 involve the use of hazardous reagents or solvents. A number of additional steps involve chromatography with its attendant high consumption of solvents and dilution of the desired products. Additionally, many of the synthetic steps disclosed in U.S. Pat. No. 5,719,020 provide very poor yields. All these factors lead to considerable difficulties and disadvantages related to implementation of commercial scale preparations of 4,7-disubstituted chromogenic derivatives of Neu5Ac. As a result, it is costly and inefficient to prepare these substances on a scale suitable for commercial development.
The present invention addresses these deficiencies. As set forth in detail herein, the number of synthetic steps and attendant number of unit operations is minimized. The use of chromatographic separations likewise is minimized. Additionally, the synthetic reactions have been optimized and the recovery procedures enhanced to provide high step yields and thereby high overall yields for the final products that are unexpected by the present state of knowledge in the field. The resulting improvements permit economical preparation of viral neuraminidase substrates on the scale of kilograms for commercial sale.